Methods for reducing gluconoylation of proteins

ABSTRACT

The present invention relates to methods of preventing glyconoylation of polypeptides produced in micororganisms.

This application is a 371 National Phase entry of internationalapplication number PCT/US04/06507, filed Mar. 4, 2004 which claimspriority to U.S. Ser. No. 60/451,686 filed Mar. 4, 2003.

FIELD OF THE INVENTION

This invention is in the field of biochemical engineering. Moreparticularly, this invention relates to fermentation processes forproducing polypeptides.

BACKGROUND OF THE INVENTION

Escherichia coli (“E. coli”) is a commonly used host for expression ofproteins for research, diagnostic, therapeutic, and industrial purposes.Modern expression systems are capable of achieving high levels of a widevariety of proteins (Baneyx, Current Opinion in Biotechnology 10:411-421(1999)). However, the quality of the expressed protein is often asimportant or more important than quantity. Proteins expressed in E. colimay be formed as insoluble aggregates, or they may have misincorporatedamino acids (Bogosian, et al., Journal of Biological Chemistry264:531-539 (1989)) or retain the N-terminal methionine (Chaudhuri, etal., Journal of Molecular Biology 285:1179-1194 (1999);Vassileva-Atanassova, et al, Journal of Biotechnology 69:63-67 (1999);Yamashita, et al., Protein Expression & Purification 16:47-52 (1999)).In addition, undesired post-translational modifications may occur suchas oxidation (Berti, et al., Protein Expression & Purification11:111-118 (1997); Konz, et al., Biotechnology Progress 14:393-409(1998)) or α-N-6-phosphogluconoylation (Geoghegan, et al., Anal.Biochem. 267:169-184 (1999); Kim et al., Acta Crystallographica SectionD-Biological Crystallography 57:759-762 (2001); Yan, et al. Biochemical& Biophysical Research Communications 262:793-800 (1999) “Yan et al. I;”Yan, et al., Biochemical & Biophysical Research Communications259:271-282 (1999) “Yan et al. II”). These modifications may adverselyeffect activity, stability, structure, or immunogenicity of theexpressed protein, greatly reducing the utility of E. coli as a host forpolypeptide expression.

Alpha(α)-N-6-phosphogluconoylation of several recombinant proteins fusedto hexahistidine affinity tags (“hexa His-tag”) has been described(Geoghegan, et al; Kim, et al.; Yan et al. I; Yan et al. II). In thesestudies, a gluconic acid derivative was found to attach to the endterminus of the recombinant protein. All of these proteins wereexpressed in B strains of E. coli using pET-based vectors (Novagen).Where reported, LB medium was used. The adduct was detected as an extramass associated with the polypeptide of either 258 Daltons (“Da”),representing the addition of 6-phosphogluconolactone (6-PGL), or 178 Da,representing the presence of gluconolactone without the phosphate.Phosphogluconoylation was presumed to occur at the N-terminal α-aminogroup through reaction with endogenous 6-PGL, an intermediate of thepentose phosphate shunt. The +178 Da adduct was proposed to be theresult of enzymatic activity acting on the +258 Da adduct to remove thephosphate. Formation of the adduct was shown to be specific to the aminoacid sequence at the N-terminus adjacent to the His-tag. Polypeptidesequences of GXXHHHH, where XX is SS, SA, AS, or AA, were the most proneto α-N-6-phosphogluconoylation, whereas SHHHHHH was less prone, andPHHHHHH and PFHHHHHH were not modified at all (Geoghegan, et al.).Modifications at other amino groups elsewhere on the protein were notdetected in vivo or in in vitro experiments that used high levels ofadded gluconolactone. (Geoghegan, et al.)

N-terminal phosphogluconoylation has been shown to inhibitcrystallization of proteins (Kim, et al.), but relatively little else isknown about its effect on protein function, stability, orimmunogenicity. It is expected that 6-PGL, being a potent electrophile,may be involved in glycation reactions in vivo (Rakitzis and Papandreou,Chemico-Biological Interactions 113:205-216 (1998)). Glycation ofproteins has been widely studied and is known to play a major role inaging and disease states related to diabetic complications (Baynes andMonnier, The Maillard Reaction in Aging, Diabetes, and Nutrition. AlanR. Liss, New York (1989)). Exogenously added delta-gluconolactone hasbeen shown to cause glycation of hemoglobin, which may be a factor inthe vascular complications of diabetes (Lindsay, et al., Clinica ChimicaActa 263:239-247 (1997)). Furthermore, glycation of alanineaminotransferase at the epsilon-amino group of Lys313 markedly reducesits catalytic activity (Beranek, et al., Molecular & CellularBiochemistry 218:35-39 (2001)).

6-Phosphogluconolactonase (“pgl”) has been shown to be an essentialenzyme of the pentose-phosphate pathway, specifically in the hydrolysisof 6-PGL to 6-phosphogluconic acid. (Miclet, et al., J. Biol. Chem.276:34840-34846 (2001)) The gene encoding this enzyme has beenidentified in human (Collard, et al., FEBS Letters 459:223-226 (1999)),Pseudomonas aeruginosa (Hager, et al., Journal of Bacteriology182:3934-3941 (2000)), and Trypanosoma brucei and Plasmodium falciparum(Miclet, et al.). Although pgl activity has long been observed in E.coli (Kupor and Fraenkel, Journal of Bacteriology 100:1296-1301 (1969)“Kupor I” and Kupor and Fraenkel, Journal of Biological Chemistry247:1904-1910 (1972) “Kupor II”), no gene sequence responsible forencoding an enzyme with this activity has been identified (Cordwell, S.J. Arch. Microbiol. 172:269-279 (1999)). It has been suggested that inaddition to enabling metabolic flux through the pentose phosphate shunt,pgl activity inside the cell prevents accumulation of 6-PGL andconsequential damaging reactions with intracellular nucleophiles(Miclet, et al.). Reported observations of phosphogluconoylation ofproteins at the N-terminus supports the hypothesis that the 6-PGLproduced in the pathway can modify proteins, but there has been noreported evidence that modulating pgl activity can affect the levels ofmodified protein.

Furthermore, Escherichia coli strain BL21 (DE3) is a commonly used hostfor expression of proteins for research, diagnostic, therapeutic, andindustrial purposes Studier, F. W., and Moffatt, B. A., J. Mol. Biol.1986 May 5; 189(1):113-130. This strain is commercially attractivebecause it achieves very high expression levels of recombinant proteinby means of coupling expression of a chromosomal copy of the T7 RNApolymerase and the use of a plasmid based T7 RNA polymerase promoter onthe recombinant protein of interest. Accordingly, since the 17 RNApolymerase is an extremely selective and active RNA polymerase,transcription of the recombinant protein accumulate to very high levels,often even to the extent that host cell transcripts are diminished.

Unfortunately, the strain BL21 (DE3) releases very low, yet detectable,infectious lambda phage particles on the order of 10-20 plaque formingunits/ml (PFU), Stewart Shuman, Proc. Natl. Acad. Sci. USA 1989;89:3489-3493. Apparently, the T7 RNA polymerase gene was introduced intothe BL21 host cell chromosome by transduction with the defective lambdaphage DE3 carrying the T7 RNA polymerase gene inserted into the lambdaint gene Studier, F. W., and Moffatt, B. A., J. Mol. Biol. 1986;189(1):113-130. The resulting defective prophage cannot replicatenormally due to the int gene interruption. Chromosomal excision of theDE3 prophage is a requirement for, the replication of the prophage priorto packaging and release of infectious phage particles and is dependanton homologous excisional recombination that is directed by the productof the int gene. As described herein very low levels of phage particlesreleased are due to abnormal, int independent random prophage excisionevents. While the release of low levels of infectious phage particlesmay be acceptable in some research laboratories, any release ofinfectious phage is totally unacceptable in the biopharmaceuticalsmanufacturing plant setting both because of considerations for patientsafety and because release of infectious agents can put at risk other E.coli based manufacturing processes.

A method for expressing or overexpressing polypeptides in amicroorganism, such as E. coli, with a reduced incidence ofphosphogluconoylation during fermentation is greatly needed. Inaddition, creation of a totally phage free BL21 (DE3) host cell would behighly desirable for the manufacture of therapeutic recombinant proteinsin the E. coli format.

SUMMARY OF THE INVENTION

The present invention provides methods for preventing gluconoylation ofpolypeptides expressed by microorganisms by growing the microorganism inrich culture medium.

The present invention also provides methods for preventinggluconoylation of polypeptides expressed by microorganisms that includeintroducing (e.g., transforming, infecting or transfecting) DNA encodinga polypeptide that demonstrates pgl activity into the microorgansim.This polypeptide may be a phosphogluconolactonase enzyme. In anotheraspect of the invention, the phosphogluconolactonase enzyme may have anamino acid sequence having at least 90% sequence identity to thephosphogluconolactonase enzyme produced by Pseudomonas, including but tolimited to, P. aeruginosa.

The present invention also provides microorganisms capable of preventinggluconoylation of proteins. In one embodiment, a microorganism maycontain DNA encoding a polypeptide that demonstrates pgl activity.

The present invention also provides an isolated polynucleotide that hasat least 90% identity to the polynucleotide set forth in SEQ ID NO:7.

The present invention also provides a microorgansim free of detectableinfectious lambda phage expression.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a restriction enzyme map of vector pECO-1-pgl-2-13.

FIG. 2 shows a polynucleotide sequence for vector pECO-1-pgl-2-13. (SEQID NO:9)

FIG. 3 shows a restriction enzyme map of vector pET28ProIL18Casp5.

FIG. 4 shows a polynucleotide sequence for vector pET28ProIL18Casp5.(SEQ ID NO:13)

FIG. 5 shows a restriction enzyme map of vector pET28proIL18casp5+pgl.

FIG. 6 shows a polynucleotide sequence for vector pET28proIL18casp5+pgl(SEQ ID NO:14).

FIG. 7 shows a polynucleotide sequence, SEQ ID NO:7.

FIG. 8 shows a polypeptide sequence, SEQ ID NO:8.

GLOSSARY

“Host cell(s)” is a cell, including but not limited to a bacterial cellor cell of a microorganism, that has been introduced (e.g., transformed,infected or transfected) or is capable of introduction (e.g.,transformation, infection or transfection) by an isolated polynucleotidesequence.

“Transformed” as known in the art, is the directed modification of anorganism's genome or episome via the introduction of external DNA orRNA, or to any other stable introduction of external DNA or RNA.

“Transfected” as known in the art, is the introduction of external DNAor RNA into a microorganism, including but not limited to recombinantDNA or RNA.

“Identity,” as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, as thecase may be, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”can be readily calculated by known methods, including but not limited tothose described in (Computational Molecular Biology, Lesk, A. M., ed.,Oxford University Press, New York, 1988; Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math.,48:1073 (1988). Methods to determine identity are designed to give thelargest match between the sequences tested. Moreover, methods todetermine identity are codified in publicly available computer programs.Computer program methods to determine identity between two sequencesinclude, but are not limited to, the GCG program package (Devereux, J.,et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, andFASTA (Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990). TheBLAST X program is publicly available from NCBI and other sources (BLASTManual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894;Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990). The well knownSmith Waterman algorithm may also be used to determine identity.

Parameters for polypeptide sequence comparison include the following:

Algorithm: Needleman and Wunsch, J. Mol. Biol. 48: 443-453 (1970)

Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl.Acad. Sci. USA. 89:10915-10919 (1992)

Gap Penalty: 12

Gap Length Penalty: 4

A program useful with these parameters is publicly available as the“gap” program from Genetics Computer Group, Madison Wis. Theaforementioned parameters are the default parameters for peptidecomparisons (along with no penalty for end gaps).

Parameters for polynucleotide comparison include the following:

Algorithm: Needleman and Wunsch, J. Mol. Biol. 48: 443-453 (1970)

Comparison matrix: matches=+10, mismatch=0

Gap Penalty: 50

Gap Length Penalty: 3

Available as: The “gap” program from Genetics Computer Group, MadisonWis. These are the default parameters for nucleic acid comparisons.

A preferred meaning for “identity” for polynucleotides and polypeptides,as the case may be, are provided in (1) and (2) below.

(1) Polynucleotide embodiments further include an isolatedpolynucleotide comprising a polynucleotide sequence having at least a70, 80, 85, 90, 95, 97 or 100% identity to the reference sequence of SEQID NO:7, wherein said polynucleotide sequence may be identical to thereference sequence of SEQ ID NO:7 or may include up to a certain integernumber of nucleotide alterations as compared to the reference sequence,wherein said alterations are selected from the group consisting of atleast one nucleotide deletion, substitution, including transition andtransversion, or insertion, and wherein said alterations may occur atthe 5′ or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among the nucleotides in the reference sequence or in oneor more contiguous groups within the reference sequence, and whereinsaid number of nucleotide alterations is determined by multiplying thetotal number of nucleotides in SEQ ID NO:7 by the integer defining thepercent identity divided by 100 and then subtracting that product fromsaid total number of nucleotides in SEQ ID NO:7, or:n _(n) ≦x _(n)−(x _(n) ·y),wherein n_(n) is the number of nucleotide alterations, x_(n) is thetotal number of nucleotides in SEQ ID NO:7, y is 0.95 for 95%, 0.97 for97% or 1.00 for 100%, and · is the symbol for the multiplicationoperator, and wherein any non-integer product of x_(n) and y is roundeddown to the nearest integer prior to subtracting it from x_(n).Alterations of a polynucleotide sequence encoding a polypeptide maycreate nonsense, missense or frameshift mutations in this codingsequence and thereby alter the polypeptide encoded by the polynucleotidefollowing such alterations.

(2) Polypeptide embodiments further include an isolated polypeptidecomprising a polypeptide having at least a 70, 80, 85, 90, 95, 97 or100% identity to a polypeptide reference sequence, wherein saidpolypeptide sequence may be identical to the reference sequence or mayinclude up to a certain integer number of amino acid alterations ascompared to the reference sequence, wherein said alterations areselected from the group consisting of at least one amino acid deletion,substitution, including conservative and non-conservative substitution,or insertion, and wherein said alterations may occur at the amino- orcarboxy-terminal positions of the reference polypeptide sequence oranywhere between those terminal positions, interspersed eitherindividually among the amino acids in the reference sequence or in oneor more contiguous groups within the reference sequence, and whereinsaid number of amino acid alterations is determined by multiplying thetotal number of amino acids by the integer defining the percent identitydivided by 100 and then subtracting that product from said total numberof amino acids, or:n _(a) ≦x _(a)−(x _(a) ·y),wherein n_(a) is the number of amino acid alterations, x_(a) is thetotal number of amino acids in the sequence, y is 0.95 for 95%, 0.97 for97% or 1.00 for 100%, and · is the symbol for the multiplicationoperator, and wherein any non-integer product of x_(a) and y is roundeddown to the nearest integer prior to subtracting it from x_(a).

“Isolated”¹ means altered “by the hand of man” from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, including but not limitedto when such polynucleotide or polypeptide is introduced back into acell.

“Polynucleotide(s)” generally refers to any polyribonucleotide orpolydeoxyribonucleotide, that may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotide(s)” include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded regions, or a mixture of single- and double-strandedregions. In addition, “polynucleotide” as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.As used herein, the term “polynucleotide(s)” also includes DNAs or RNAsas described above that comprise one or more modified bases. Thus, DNAsor RNAs with backbones modified for stability or for other reasons are“polynucleotide(s)” as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein. It will be appreciated that a great variety ofmodifications have been made to DNA and RNA that serve many usefulpurposes known to those of skill in the art. The term“polynucleotide(s)” as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including, for example, simple and complex cells.“Polynucleotide(s)” also embraces short polynucleotides often referredto as oligonucleotide(s).

“Polypeptide(s)” refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds. “Polypeptide(s)” refers to both short chains, commonly referredto as peptides, oligopeptides and oligomers and to longer chainsgenerally referred to as proteins. Polypeptides may comprise amino acidsother than the 20 gene encoded amino acids. “Polypeptide(s)” includethose modified either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques. Such modifications are well described in basic texts and inmore detailed monographs, as well as in a voluminous researchliterature, and they are well known to those of skill in the art. Itwill be appreciated that the same type of modification may be present inthe same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may comprise many types of modifications.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains, and the amino or carboxyl termini.Modifications include, for example, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, proteolyticprocessing, phosphorylation, prenylation, racemization, glycosylation,lipid attachment, sulfation, gamma-carboxylation of glutamic acidresidues, hydroxylation and ADP-ribosylation, selenoylation, sulfation,transfer-RNA mediated addition of amino acids to proteins, such asarginylation, and ubiquitination. See, for instance, PROTEINS—STRUCTUREAND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman andCompany, New York (1993) and Wold, F., Posttranslational ProteinModifications: Perspectives and Prospects, pgs. 1-12 inPOSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed.,Academic Press, New York (1983); Seifter et al., Meth. Enzymol.182:626-646 (1990) and Rattan et al., Protein SynthesisPosttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). Polypeptides may be branched or cyclic, with or withoutbranching. Cyclic, branched and branched circular polypeptides mayresult from post-translational natural processes and may be made byentirely synthetic methods, as well.

“Recombinant expression system(s)” refers to expression systems orportions thereof or polynucleotides of the invention introduced ortransformed into a host cell or host cell lysate for the production ofthe polynucleotides and polypeptides of the invention.

“Variant(s)” as the term is used herein, is a polynucleotide, orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusion proteins and truncations inthe polypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. The present invention also includes include variants of each ofthe polypeptides of the invention, that is polypeptides that vary fromthe referents by conservative amino acid substitutions, whereby aresidue is substituted by another with like characteristics. Typicalsuch substitutions are among Ala, Val, Leu and Ile; among Ser and Thr;among the acidic residues Asp and Glu; among Asn and Gln; and among thebasic residues Lys and Arg; or aromatic residues Phe and Tyr.Particularly preferred are variants in which several, 5-10, 1-5, 1-3,1-2 or 1 amino acids are substituted, deleted, or added in anycombination. A variant of a polynucleotide or polypeptide may be anaturally occurring such as an allelic variant, or it may be a variantthat is not known to occur naturally. Non-naturally occurring variantsof polynucleotides and polypeptides may be made by mutagenesistechniques, by direct synthesis, and by other recombinant methods knownto skilled artisans.

“Microorganism(s)” means a (i) prokaryote, including but not limited to,a member of the genus Streptococcus, Staphylococcus, Bordetella,Corynebacterium, Mycobacterium, Neisseria, Haemophilus, Actinomycetes,Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella,Pasturella, Moraxella, Acinetobacter, Erysipelothrix, Branhamella,Actinobacillus, Streptobacillus, Listeria, Calymmatobacterium, Brucella,Bacillus, Clostridium, Treponema, Escherichia, Salmonella, Kleibsiella,Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillum,Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia,Chlamydia, Borrelia and Mycoplasma, and further including, but notlimited to, a member of the species or group, Group A Streptococcus,Group B Streptococcus, Group C Streptococcus, Group D Streptococcus,Group G Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes,Streptococcus agalactiae, Streptococcus faecalis, Streptococcus faecium,Streptococcus durans, Neisseria gonorrheae, Neisseria meningitidis,Staphylococcus aureus, Staphylococcus epidermidis, Corynebacteriumdiptheriae, Gardnerella vaginalis, Mycobacterium tuberculosis,Mycobacterium bovis, Mycobacterium ulcerans, Mycobacterium leprae,Actinomyctes israeli, Listeria monocytogenes, Bordetella pertusis,Bordatella parapertusis, Bordetella bronchiseptica, Escherichia coli,Shigella dysenteriae, Haemophilus influenzae, Haemophilus aegyptius,Haemophilus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonellatyphi, Citrobacter freundii, Proteus mirabills, Proteus vulgaris,Yersinia pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratialiquefaciens, Vibrio cholera, Shigella dysenterii, Shigella flexneri,Pseudomonas aeruginosa, Franscisella tularensis, Brucella abortis,Bacillus anthracis, Bacillus cereus, Clostridium perfringens,Clostridium tetani, Clostridium botulinum, Treponema pallidum,Rickettsia rickettsii and Chlamydia trachomitis, (ii) an archaeon,including but not limited to Archaebacter, and (iii) a unicellular orfilamentous eukaryote, including but not limited to, a protozoan, afungus, a member of the genus Saccharomyces, Kluveromyces, or Candida,and a member of the species Saccharomyces ceriviseae, Kluveromyceslactis, or Candida albicans.

“Bacteria(um)(I)” means a (i) prokaryote, including but not limited to,a member of the genus Streptococcus, Staphylococcus, Bordetella,Corynebacterium, Mycobacterium, Neisseria, Haemophilus, Actinomycetes,Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella,Pasturella, Moraxella, Acinetobacter, Erysipelothrix, Branhamella,Actinobacillus, Streptobacillus, Listeria, Calymmatobacterium, Brucella,Bacillus, Clostridium, Treponema, Escherichia, Salmonella, Kleibsiella,Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillum,Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia,Chlamydia, Borrelia and Mycoplasma, and further including, but notlimited to, a member of the species or group, Group A Streptococcus,Group B Streptococcus, Group C Streptococcus, Group D Streptococcus,Group G Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes,Streptococcus agalactiae, Streptococcus faecalis, Streptococcus faecium,Streptococcus durans, Neisseria gonorrheae, Neisseria meningitidis,Staphylococcus aureus, Staphylococcus epidermidis, Corynebacteriumdiptheriae, Gardnerella vaginalis, Mycobacterium tuberculosis,Mycobacterium bovis, Mycobacterium ulcerans, Mycobacterium leprae,Actinomyctes israelii, Listeria monocytogenes, Bordetella pertusis,Bordatella parapertusis, Bordetella bronchiseptica, Escherichia coli,Shigella dysenteriae, Haemophilus influenzae, Haemophilus aegyptius,Haemophilus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonellatyphi, Citrobacter freundii, Proteus mirabilis, Proteus vulgaris,Yersinia pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratialiquefaciens, Vibrio cholera, Shigella dysenterii, Shigella flexneri,Pseudomonas aeruginosa, Franscisella tularensis, Brucella abortis,Bacillus anthracis, Bacillus cereus, Clostridium perfringens,Clostridium tetani, Clostridium botulinum, Treponema pallidum,Rickettsia rickettsii and Chlamydia trachomitis, and (ii) an archaeon,including but not limited to Archaebacter.

As used herein, “heterologous polypeptide(s)” refers to a polypeptidenot naturally synthesized by a transformed host cell or microorganism ofinterest and introduced into the host cell or microorganism byrecombinant DNA. For example, E. coli may act as a host microorganismfor the expression of interleukin, which does not occur innon-transformed E. coli. Heterologous polypeptides may includepolypeptides that have been modified to facilitate isolation.

As used herein “affinity tag” refers to any moiety associated with amolecule that may give said molecule a selective affinity for anothersubstance or molecule. For instance, an affinity tag may be used tofacilitate purification of a molecule by providing the molecule with aselective affinity for a column's packing material. A non-limitingexample of an affinity tag is a his-tag.

As used herein, “His-tag” refers to a repeat of histidine amino acidsand may include other amino acids encoded into a polypeptide throughengineering techniques. Typically, a His-tag contains at least six(“hexa”) histidine repeats and is located near the N-terminus of thepolypeptide. His-tags can be used to facilitate purification ofheterologous polypeptides over Ni-columns.

As used herein, “minimal medium” refers to cell growth medium comprisingchemically defined ingredients including, but not limited to, bufferssuch as phosphate buffer, salts such as magnesium sulfate, calciumchloride, sodium chloride, or manganese sulfate, minerals such as iron,zinc, copper, cobalt, or molybendum, a carbon source such as glucose orglycerol, and a nitrogen source such as ammonium sulfate or nitrate. Anexample is M9 media (Kim, Y S, J H Seo and H Y Cha, Enzyme and MicrobialTechnology 33 (2003):460-465.)

As used herein, “rich medium” refers to cell growth medium comprisingundefined ingredients and including, but not limited to, additionalcomponents such as buffers (for example phosphate buffer), salts (forexample magnesium sulfate, calcium chloride, sodium chloride, ormanganese sulfate), and a carbon source which may be glucose orglycerol, for example. The nitrogen source may be comprised of a proteinhydrolysate such as yeast extract, meat hydrolysate, or soy hydrolysate.The nitrogen source may be provided at a concentration capable ofsupporting cell growth at a ratio of complex nitrogen source (in gramsper liter) to maximum cell density (in OD units) of 1:1 or greater.Examples of rich medium include, but are not limited to, Superbroth(Atlas R M, Handbook of Microbiological Mesdia; Parks L C Ed.; CRCPress: Boca Raton Fla., pp. 281, 523, 529, 859), Teriffic Broth (AlphaBiosciences, Baltimore, Md. USA), Turbo Broth (Athena Enzyme Systems,Baltimore, Md. USA), or Hyper Broth (Athena Enzyme Systems, Baltimore,Md. USA).

As used herein, “gluconoylation” refers to the attachment of a gluconicacid derivative to a protein. Gluconoylation may include, but is notlimited to, 6-phosphogluconolactone (6-PGL) adduct formation,acetylation, formylation, deformylation, gluconolactonation, or gluconicacid derivatization.

As used herein, “titer yield” refers to the concentration of a product(e.g., heterologously expressed polypeptide) in solution (e.g., culturebroth or cell-lysis mixture or buffer) and it usually expressed as mg/Lor g/L. An increase in titer yield may refer to an absolute or relativeincrease in the concentration of a product produced under two definedset of conditions.

As used herein, “pgl activity” refers to any activity of a6-Phosphogluconolactonase (“pgl”). Such activity may include hydrolysisof 6-PGL to 6-phosphogluconic acid. Significant pgl activity may bedefined as hydrolysis activity of at least 0.2 IU/min/g.

As herein used, the terms “stringent conditions” and a “stringenthybridization conditions” mean hybridization will occur only if there isat least 70% and preferably at least 80%, but especially preferably atleast 95% identity between the sequences. An example of stringenthybridization conditions is overnight incubation at 42° C. in a solutioncomprising: 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 micrograms/ml denatured, sheared salmon sperm DNA,followed by washing the filters in 0.1×SSC at about 65° C. Hybridizationand wash conditions are well known and exemplified in Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y., (1989), particularly Chapter 11 therein, the disclosure ofwhich is hereby incorporated in its entirety by reference.

DETAILED DESCRIPTION OF THE INVENTION

Cellular stresses arising from growth on minimal medium, high celldensities, and high-level heterologous polypeptide expression, amongother stresses, may contribute to the gluconoylation of heterologouspolypeptide. Alleviating one or more of these stressors may thereforeaffect the level of gluconoylation of protein expressed in themicroorganism, which in turn may effect the purity and titer yield of adesired polypeptide. Some strains of E. coli may have higher levels ofpgl activity than others (although the gene responsible for thisactivity has not been identified in E. coli. Cordwell, S. J. 1999. Arch.Microbiol. 172:269-279. Furthermore, gluconoylation of polypeptides mayaffect crystallization and structural determination of thesepolypeptides.

The present invention provides methods for preventing gluconoylation ofa polypeptide expressed in a microorganism, comprising growing saidmicroorganism in rich culture medium. In another aspect of theinvention, the microorganism does not demonstrate significant6-phosphogluconolactonase activity when grown in minimal medium. Themicroorganism may be a strain of E. coli. In another aspect of theinvention, an E. coli is a B strain. In another aspect of the invention,a rich culture medium comprises a complex nitrogen source. A richculture medium is capable of maintaining cell growth at ratio of 1:1 forconcentration of complex nitrogen source to cell density. A complexnitrogen source may comprise tryptone, peptone, or yeast extract. Inanother aspect of the invention, a culture medium is Superbroth.Superbroth medium may be doubly concentrated. In another aspect of theinvention, a microorganism is transfected with a recombinant DNAmolecule encoding a heterologous polypeptide.

In another embodiment of the present invention, methods are provided forpreventing gluconoylation of a polypeptide expressed in E. colicomprising fermenting a K strain variety of E. coli for the expressionof the polypeptide. In another aspect of the invention, a K straindemonstrates at least about 100-fold higher phosphogluconolactonaseactivity compared with B strain grown under substantially the sameconditions. In another aspect of the invention, a K strain is K-12.

In another embodiment of the present invention, methods are provided forpreventing gluconoylation of polypeptides expressed in a microorganism,comprising introducing DNA into the microorganism, wherein the DNAencodes a polypeptide that demonstrates 6-phosphogluconolactonaseactivity. In another aspect of the invention, a microorgansim is E.coli. A DNA comprises DNA encoding a 6-phosphogluconolactonase enzyme.In another aspect of the invention, a DNA encoding a6-phosphogluconolactonase enzyme has at least 70%, 80%, 85%, 90%, 95%,97%, or 100% sequence identity to a DNA encoding6-phosphogluconolactonase from a Pseudomonas, which may be P.aeruginosa. In another aspect of the invention, a DNA encoding a6-phosphogluconolactonase enzyme has at least 70%, 80%, 85%, 90%, 95%,97%, or 100% sequence identity to SEQ ID NO:7. In another aspect of theinvention, a 6-phosphogluconolactonase enzyme has an amino acid sequencehaving at least 70%, 80%, 85%, 90%, 95%, 97%, or 100% sequence identityto SEQ ID NO:8. A DNA encoding a 6-phosphogluconolactonase enzymecomprises the sequence set forth in SEQ ID NO:7. DNA may be recombinantDNA, and/or it may be transformed into a genomic DNA of a microorganism.

In another embodiment of the present invention, a microorganism isprovided that is capable of preventing gluconoylation of polypeptides. Amicroorganism comprises DNA that encodes a polypeptide that demonstrates6-phosphogluconolactonase activity. A microorganism comprises DNA thatencodes 6-phosphogluconolactonase. In another aspect of the invention, aDNA that encodes 6-phosphogluconolactonase has at least 70%, 80%, 85%,90%, 95%, 97%, or 100% sequence identity to DNA encoding a6-phosphogluconolactonase enzyme from a Pseudomonas and wherein themicroorgansim is not Pseudomonas. In another aspect of the invention, aDNA that encodes 6-phosphogluconolactonase is from P. aeruginosa. Inanother aspect of the invention, a microorganism is a strain of E. coli.In another aspect of the invention, a microorganism is a strain of E.coli and the wherein a DNA that encodes 6-phosphogluconolactonase isincorporated into the genome of the microorganism.

In another embodiment of the present invention, an isolatedpolynucleotide is provided comprising a polynucleotide having at least a70%, 80%, 85%, 90%, 95%, 97%, or 100% identity to the polynucleotide setforth in SEQ ID NO:7. In another aspect of the invention, an isolatedpolynucleotide comprises a polynucleotide having at least a 70%, 80%,85%, 90%, 95%, 97%, or 100% sequence identity to a polynucleotideencoding a polypeptide comprising amino acids having at least 70%, 80%,85%, 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO:8. In anotheraspect-of the invention, a DNA encodes a protein demonstrating6-phosphogluconolactonase activity. In another aspect of the invention,a DNA sequence is set forth in SEQ ID NO:7. In another aspect of theinvention, a DNA sequence is homologous to a DNA encoding6-phosphogluconolactonase from P. aeruginosa. In another aspect of theinvention, an isolated polynucleotide further comprises a polynucleotidethat encodes a human interleukin-18, or variant thereof.

In another embodiment of the present invention, methods are provided forimproving polypeptide crystal formation comprising expressing saidpolypeptide by a method of the invention in order to reduceglyconoylation of the polypeptide.

In another embodiment of the present invention, methods are provided forimproving growth efficiency and expression of a polypeptide in amicroorganism. In one aspect, a polypeptide demonstrates an increasedtiter yield at fermentation stage compared with gluconoylatedpolypeptide.

In another embodiment of the present invention, methods are provided forproducing a human interleukin comprising co-expressing said humaninterleukin in a microorganism with an enzyme having6-Phosphogluconolactonase (“pgl”) activity. In one aspect, a humaninterleukin is an IL-18 protein, or variant thereof. In one aspect, amicroorgansim is E. coli. In one aspect, the pgl is encoded by apolynucleotide that is transformed or transfected into the E. coli,including but not limited to being transformed into the genome orepisome.

In another embodiment of the present invention, methods are providedproducing a human interleukin comprising expressing the humaninterleukin in a microorganism that is grown in a rich culture medium.In one aspect, a human interleukin is IL-18 protein, or a variantthereof. In one aspect, the microorgansim is E. coli. In one aspect, arich culture medium comprises phytone peptone. In one aspect, a richculture medium comprises bacto yeast extract.

In another embodiment of the present invention, a microorgansim free ofdetectable infectious lambda phage expression is provided. In oneaspect, lambda phage capsid structural protein, gpE, is deleted ordisrupted within a microorganism. Deletion or disruption of a proteincan be achieved in several non-limiting ways. A gene encoding a proteinmay be either partially or completely removed from a microorganism. Agene encoding a protein can be genetically modified by such non-limitingtechniques as point mutation or random mutation. A protein may becleaved or modified chemically or enzymatically to disrupt its structureor function. A promoter regulating gene expression can be disrupted ordeleted to eliminate or reduce gene expression. Solution conditions ortemperature can be regulated to effect protein structure or function. Inone aspect, a microorganism is an E. coli. In another aspect amicroorganism comprises a T7 RNA polymerase gene.

The following examples illustrate various aspects of this invention.These examples do not limit the scope of this invention which is definedby the appended claims.

EXAMPLES Example 1

An improved strain of E. coli BL21 (DE3) strain was created that is freeof detectable infectious lambda phage expression by knocking out a majorlambda phage capsid structural protein gpE. This strain was used in theExamples presented herein.

A 413 base pair internal fragment of the gpE coding region was PCRamplified using the primer pair:

5′ AGCTGGCCATTGCTCAGGTCGAAG 3′ (SEQ ID NO: 1) 5′GTACTGTCCGGAATACACGACGATG 3′ (SEQ ID NO: 2)and BL21 (DE3) chromosomal DNA as template. The resulting fragment:

(SEQ ID NO: 3) AGCTGGCCAT TGCTCAGGTC GAAGAGATGC AGGCAGTTTCTGCCGTGCTT AAGGGCAAAT ACACCATGAC CGGTGAAGCCTTCGATCCGG TTGAGGTGGA TATGGGCCGC AGTGAGGAGAATAACATCAC GCAGTCCGGC GGCACGGAGT GGAGCAAGCGTGACAAGTCC ACGTATGACC CGACCGACGA TATCGAAGCCTACGCGCTGA ACGCCAGCGG TGTGGTGAAT ATCATCGTGTTCGATCCGAA AGGCTGGGCG CTGTTCCGTT CCTTCAAAGCCGTCAAGGAG AAGCTGGATA CCCGTCGTGG CTCTAATTCCGAGCTGGAGA CAGCGGTGAA AGACCTGGGC AAAGCGGTGTCCTATAAGGG GATGTATGGC GATGTGGCCA TCGTCGTGTA TTCCGGACAG TACPCR amplification product was TA cloned and sequence verified usingstandard techniques. Next, a functional copy of a chloramphenicolresistance gene was introduced into the EcoRV site centrally located andshown bolded in the cloned gpE sequence. The chloramphenicol interruptedgpE fragment construct was then used as a linear homologousrecombination knock-out vector for the genetic knock-out of the lambda(DE3) gpE gene residing in E. coli host strain BL21 (DE3). Thisknock-out vector was introduced into the host cell exactly as describedby Kirill A. Datsenko and Barry Wanner, Proc. Natl. Acad. Sci. USA 2000;97:6640-6645, except the vector described herein was used. Putative gpEknock-outs were preliminarily identified by the ability to grow in thepresence of 6 μg/ml chloramphenicol, and were subsequently confirmed byPCR analysis. One such gpE knock-out of BL21 (DE3) was banked and giventhe GSK designation ECC-023.

ECC-023 was tested for the presence of infectious phage particles in thevery sensitive plaque assay. As shown in Table 1, the parental BL21(DE3) expressed barely detectable amounts of phage when not induced todo so, approximately 20 pfu, but nearly three orders of magnitude morewhen induced by the SOS response to genetic damage (UV irradiation). ThegpE knock-out derivative ECC-023 however did not produce any evidence ofinfectious phage, even when treated to induce the SOS response. The datashows that the BL21 (DE3) derivative ECC-023 does not produce anydetectable infectious phage particles and accordingly is a suitable hostcell strain for biopharmaceutical manufacturing.

TABLE 1 Production of infectious phase particles from E. coli. PFU/mlNot Induced SOS Induced BL21(DE3) 21 10,000 ECC-023 0 0

Example 2

This example shows that culturing cells in a rich medium can preventformation of a 6-PGL adduct of His-tagged proteins as compared tominimal medium.

E. coli BL21 (DE3) transformed with plasmid pET28FabZSa, which encodesthe gene for Hexa-his-FabZ, was grown in MCJK (minimal medium) or MCJKLB(rich medium) at 37° C. until an OD₆₀₀ of 1.0 was reached. Mediumcompositions for MCJK and MCJKLB are described in Table 2.

TABLE 2 Medium compositions for production of Hexahis-FabZ ComponentMCJK MCJKLB K₂HPO₄ 56 mM 56 mM (NH₄)₂SO₄ 38 mM 38 mM MgSO₄ 5 mM 5 mMCaCl₂ 1 mM 1 mM Trace metals* 1 mL/L 1 mL/L Glucose 10 g/L 10 g/L Yeastextract none 5 g/L Bacto-tryptone none 10 g/L *Trace metals: 161 mMFeCl₂, 2.7 mM Na₂MoO₄, 1.4 mM ZnSO₄, 2.5 mM MnCl₂, 3.2 mM CuSO₄, 3.4 mMCoCl₂, and 3.2 mM H₃BO₃

Isopropyl-beta-D-thiogalactopyranoside (“IPTG”) was added to a finalconcentration of 1 mM and the cells were maintained at 37° C. for fourhours until harvest. Cells were collected by centrifugation at 16,000×gfor 10-min and lysed by gentle agitation in 10 mM Tris, pH 8.0, 1 mMNa₂EDTA, 10 μg/mL lysozyme and 25 U/mL Benzonase (Sigma). Cell debriswas removed by centrifugation at 16,000×g for 10 minutes and therecombinant protein was purified from the soluble extract with Ni-NTAresin in batch mode according to the manufacturer's recommendations(Qiagen Inc., Valencia, Calif.). Heterologous proteins were analyzed forthe presence of the gluconolactone modification by LC/MS analysis.

MGSSH₄ His-tag heterologous polypeptide is gluconoylated at a level of34% of total recombinant protein when the culture is performed inminimal medium (MCJK) as shown in Table 3. When the medium issupplemented or made rich with yeast extract and tryptone, gluconoylatedprotein is not detectable, also shown in Table 3. Thus, the use of aricher medium can prevent formation of the 6-PGL adduct.

TABLE 3 Effect of minimal vs. rich medium on gluconoylation of a His-tagged protein expressed in E. coli Vector N-Terminus Insert Modified %Host Medium Comments pET28FabZSa MGSSH₆ FabZ 34% BL21(DE3) MCJK minimalmedium pET28FabZSa MGSSH₆ FabZ Not BL21(DE3) MCJKLB rich mediumdetectable

Example 3

This example demonstrates that an internal lysine residue of arecombinant CXC chemokine (otherwise known as Gro-beta-T) can becomegluconoylated when expressed in E. coli. The cDNA and amino acidsequences of human Gro-beta-T are provided in International PatentApplication, Publication No. WO 92/00327 (Jan. 9, 1992) as well as U.S.Pat. No. 6,042,821 and U.S. Pat. No. 6,413,510, incorporated herein byreference in their entirety.

Heterologous protein accumulation during production of a CXC chemokine(Gro-beta-T) was routinely monitored using a quantitative, highthroughput reversed phase assay. This separation was interfaced with ahigh-performance electrospray time-of-flight (“ESI-TOF”) massspectrometer to provide a means of routinely monitoring formation ofproduct related variants.

When expressed in the E. coli BL21 (DE3) host, two unique productmodifications were observed. Deconvolution of the mass spectracharacterized the variants as exhibiting mass shifts of +178 Da and +258Da compared to native product at 7,542 Da.

Product derived from these fermentations was passed through apurification procedure. In-process samples were characterized using ahigh resolution reversed phase assay that is capable of resolving minorproduct variants. Liquid chromatography mass spectroscopy (“LCMS”)analysis confirmed that the new product variants exhibited mass shiftsof +258 Da (ret. time ˜14.8 min) and +178 Da (ret. time ˜15.2 Da).

Published literature indicated that these +258 Da and +178 Da massshifts had previously been observed in proteins expressed with hexaHis-tags resulting from the addition of 6-PGL or gluconolactone to theN-terminal residue. The CXC chemokine (Gro-beta-T) was not expressedusing a hexa His-tags. Furthermore, the molecule does not exhibit anyhistidine rich regions. To confirm that the species were productrelated, each variant was isolated and subjected to N-terminalsequencing. Sequencing confirmed proper product sequence and ensuredthat no hexa His-tag was present.

Variants were subjected to trypsin digestion and peptide mapping.Comparison with peptide maps generated using product standard revealedtwo new peaks in the tryptic map with the first peak eluting at 45.8 min(m/z 1094) and the second peak eluting at 46.5 min (m/z 1174). The newpeptides were isolated and subjected to N-terminal sequencing whichindicated that both had the same sequence (NIQSVKVK (SEQ ID NO:4)). Thesequence homology and presence of a 80 Da mass difference suggested thatthe species differed only by the presence of phosphorylation.

LCMS mass spectrometry analysis was performed on the peak with m/z 1174.The MS/MS experiment confirmed the peak has a sequence of NIQSVKVK (SEQID NO:4). In addition, the y series fragment ions starting from y3including y3, y4, y5, and y6 shown mass increase of 258 Da. However, they2 ion did not show mass increase of 258 Da. This data indicated thatthe modification was on Lysine (no. 23 in the sequence) with massincrease of 258 Da.

To confirm that the modification observed was due to productgluconoylation, in vitro reactions were developed that employed reactiveL-glucono-1,5-lactone or galactonic acid. Previous work had shown thatthese species were capable of inducing addition of gluconoyl species tothe hexa His-tag [Geohegan, et al.].

Purified CXC chemokine (Gro-beta-T) was buffer exchanged into a TRISbuffer at pH 8.0. L-glucono-1,5-lactone or galactonic acid were added toa concentration of 450 mM and samples were incubated at room temperaturefor twenty minutes. Both lactones induced the same +178 Da modificationof the CXC chemokine observed during expression in E. coli.

Example 4

This example illustrates the gluconoylation of internal lysine residuesof a recombinant human interleukin (human IL-18) expressed in E. coli.

High resolution reversed phase HPLC coupled with ESI-TOF massspectrometry was employed to screen an interleukin product for 6-PGLadducts. Analysis of B-strain expressed interleukin yielded a productrelated species exhibiting a mass difference of +178 Da. Since theprotein was expressed without a hexa His-tag, the modification waslikely occurring at an internal lysine residue as had been observed withthe CXC chemokine (see Example 3).

High resolution reverse phase HPLC analysis of the interleukin moleculeenabled identification of multiple product species exhibiting +178 Damass shifts at RT 21.0, 22.0, 23.0 minutes and an additional minorspecies exhibiting a +258 Da shift (RT ˜27.5 minutes).

In vitro modification of the interleukin was also evaluated withL-glucono-1,5-lactone at pH 8.0, as described in the previous example.This reaction again yielded the same +178 Da modifications observed whenthe interleukin was expressed in B strain of E. coli.

Example 5

This example shows that culturing cells in rich medium preventsgluconoylation or PGL adduct formation of internal lysine residues of arecombinant protein. Three fed-batch fermentation processes werecompared in this study. The difference between the fermentation runs wasthe batch medium and feed compositions used during the production phase.A rich medium was used in the Process I fermentation, while the secondand third batches (Processes II and III) were performed with a minimalmedium. Glycerol was used as feed medium in Process I and II, whileglucose was used in Process III.

The fermentation process to produce a recombinant interleukin in E. coliwas carried out in a fed-batch mode in a 15-liter fermenter. Thefermentation was started as a batch process. The inoculum was preparedby aseptically transferring 0.5 mL of a glycerol stock culture into 1000ml of PYE medium in a 2.8-L flask, as shown in Table 4.

TABLE 4 Seed medium composition for expansion of inoculum intofermenters. Batch Medium Item concentration Yeast Extract 5 g/L Phytonepeptone 10 g/L Sodium chloride 10 g/L Dextrose 10 g/L Kanamycin sulfate50 mg/L

The culture was grown overnight at 27.5° C. on a rotary shaker. Theshake flask culture was then transferred into a fermenter containing 5 Lof batch medium. The production medium consists of salts, amino acids,and kanamycin sulfate. Additionally, the rich medium is supplementedwith high concentrations of yeast extract and phytone peptone, as shownin Table 5.

TABLE 5 Production stage medium composition for comparison of rich vs.minimal medium. Batch Medium Concentration Item Process I Process IIProcess III Yeast Extract 48 g/L 5.8 g/L 5.8 g/L Phytone peptone 24 g/L1.6 g/L 1.6 g/L (NH₄)₂SO₄ none 5 g/L 5 g/L NaCl none 1.3 g/L 1.3 g/LDextrose none none 33.5 g/L Glycerol 26 g/L 26 g/L none K₂HPO₄ 15.3 g/L6 g/L 6 g/L KH₂PO₄ 1.7 g/L 3 g/L 3 g/L Trace metals* 1 mL/L 1 mL/L 1mL/L MgSO₄ 5 mM 5 mM 5 mM CaCl₂ 1 mM 1 nM 1 nM Kanamycin sulfate 30 mg/L30 mg/L 30 mg/L *Trace Metals solution: FeCl₂•6H₂O, 37.8 g/L;Na₂MoO₄•H₂O, 0.7 g/L; ZnSO₄•7H₂O, 0.4 g/L; MnCl₂•4H₂O, 0.5 g/L;CuSO₄•5H₂O, 0.8 g/L; CoCl₂•6H₂O, 0.8 g/L; H₃BO₃, 0.2 g/L.

The pH of each medium was maintained at approximately 7.3 using ammoniumhydroxide. Temperature was controlled at about 27.5° C. Feed medium,glycerol or dextrose was sterilized separately and aseptically connectedto a fermenter using silicone rubber tubing.

Supply of feed medium was initiated when the initial batch amount inculture was depleted. A polarographic oxygen electrode monitoreddissolved oxygen tension in the fermenter. Carbon feed rate wascontrolled in a cascade mode to keep constant dissolved oxygen tensionabove 20%. Aeration was maintained at 10 slpm and pressure at 7.0 psigover pressure. Agitation speed of 800 rpm was used from beginning to theend of the fermentation run. At an OD₅₅₀ of approximately 40 units, 100mL of 1 mM IPTG was injected into culture to induce recombinant geneexpression.

Reverse-phase HPLC analysis of the samples collected 24 hours post IPTGaddition indicated reduction in the level of gluconolactone in theproduct. The absence of 6-PGL adduct in Process I, and its presence inProcess II and III, indicates that the use of rich medium, and notglycerol carbon source, was responsible for elimination of the 6-PGLadduct. Table 6 summarizes the results of the analysis.

TABLE 6 Analysis of phosphogluconolactone adduct in three fermentationprocesses to produce a recombinant interleukin. phosphogluconolactoneadduct on human Process No. interluekin (IL-18) Process I Absent ProcessII Present Process III Present

Example 6

This example shows that the K-12 strain of E. coli has 100-fold higherpgl activity than B strain.

6-Phosphogluconolactonase activity was measured in extracts of twodifferent strains with BL12(DE3) as the parental one, ECC005 and ECO680,and two other strains originated from a K-12 genotype, ECO706 andATG3995.

To culture the strains, 2-L shake flasks containing 1 L of PYE medium+1% glucose and 50 μg/ml kanamycin were inoculated with 0.4 mL of cellsuspension from frozen vials. Incubation was at 30° C., with anagitation of 220 rpm for 6 or 7.5 hours. Samples were taken at theindicated time points to measure the optical density at 550 nm andcollect pellets to measure the pgl activity. Details of in vitro assayto measure the pgl activity is presented below.

The pgl activity was assayed fluorimetrically, at 340 nm and 25° C., incell-free extracts. The cell-free extracts were prepared as previouslydescribed (Maitra and Lobo, Journal of Biological Chemistry 246: 475-488(1971)); with essentially the same resuspension buffers, 50 mM-HEPES pH7.3 containing 5 mM-EDTA and 5 mM-β-mercaptoethanol, but cells werebroken by sonication (5 cycles of 20 sec.).

The pgl was assayed using a previously published method (Sinha andMaitra Journal of General Microbiology, 1992; 138: 1865-1873) with somemodifications. The assay consisted of a pre-incubation of 1 mL-mixturecontaining 50 μM-glucose-6-phosphate, 0.5 mM-NADP+, and 1 unit ofglucose-6-phosphate dehydrogenase in 100 mM-MES buffer, pH 6.5,containing 25 mM-KCl and 10 mM-MgCl2. Once the reaction reached theplateau, it was followed by the addition of 2 units of6-phosphogluconate dehydrogenase resulting in a slow increase in theabsorbance due to the spontaneous hydrolysis of the 6-PGL formed duringthe pre-incubation step. At the addition of cell-free extracts, anysignificant increment in the fluorescence is because of thephosphoglunolactonase activity. All pgl activities were estimated in thelinear range of the absorbance increase following addition of thecell-free extract.

The enzyme activity was estimated by subtracting the rate of spontaneoushydrolysis of 6-PGL. The enzyme activities were expressed in mmol NADPHper minute and per mg of total protein. Total protein was determined bythe Bicinchoninic acid (BCA) Protein Assay (Pierce, Ill., USA).

In the strains based parentally in a K-12 genotype, we have detectedupwards of 100-fold more activity than in B strains.

Table 7 summarizes the results of the enzyme assays on the two strainbackgrounds.

TABLE 7 Phosphogluconolactonase activity measured in K-12 and B strainsof E. coli Activity Sp. Hydrolysis Cultivation (mmol NADH/ (mmol/min/mgStrain Time (h) min/mg protein) protein) “K” STRAINS ATCC 10798 5.51.061 0.0011 6.5 2.899 0.0012 7.5 2.584 0.0012 K-12 expressing a 7.53.539 0.0011 human interleukin (IL-18) “B” STRAINS ATCC 47092 5.5 0.0280.0012 BL21(DE3) expressing 5.5 0.032 0.0012 a human interleukin (IL-18)6.5 0.019 0.0011 7.5 0.032 0.0012

Example 7

This example shows that expressing a heterologous polypeptide, a CXCchemokine (otherwise known as Gro-beta-T), in E. coli BL21 strainresults in 6-PGL adduct, whereas expressing the same polypeptide in aK-12 strain prevents gluconoylation of internal lysines.

Ten fermentation runs were carried out using four different strains ofE. coli constructed to recombinantly express a CXC chemokine. Thisexample demonstrates the effect of host background versus promotersystem on the presence of 6-PGL adduct formation of the recombinantprotein by using two expression systems with each host cell background.Table 8 describes the strains that were used.

TABLE 8 Host background and promoter systems used to express a CXCchemokine in E. coli. Strain No. Host Background Promoter system 1 BL21(DE3) T7 polymerase 2 K-12 (DE3) T7 polymerase 3 BL21 (DE3) Lambda pL 4K-12 Lambda pL

Fermentation to produce the CXC chemokine (Gro-beta-T) in E. coli wascarried out in a fed-batch mode in a 15-liter fermenter. Fermentationwas started as a batch process. Inoculum was prepared by asepticallytransferring 0.75 mL of a glycerol stock culture into 100 mL of PYEmedium in a 500 mL baffled flask. Culture was grown for 7 hours at 32.0°C. on a rotary shaker. Shaken flask culture was then mixed with 1.0 L ofPYE medium and transferred into a fermenter containing 8.0 L of batchmedium. Production medium consisted of salts and either carbenicillin orkanamycin sulfate depending on the promoter. Media formulations for seedexpansion are provided in Table 9, and medium formulation for productionof CXC chemokine in E. coli are provided in Table 10.

TABLE 9 Medium formulation for seed expansion. Component ConcentrationYeast Extract 5 g/L Phytone peptone 10 g/L Sodium chloride 10 g/LDextrose 10 g/L Kanamycin sulfate 50 mg/L

TABLE 10 Medium formulation for production of CXC chemokine in E. coliComponent Concentration Ammonium Sulfate 5 g/L Potassium phosphate,Dibasic 6 g/L Potassium phosphate, Monobasic 3 g/L Sodium chloride 1.3g/L Dextrose 18 g/L Magnesium sulfate 4.8 g/L Biotin 1 mg/L TraceMetals* 10 mL/L Kanamycin sulfate 50 mg/L -or- Carbenicillin 25 mg/L*Trace Metals solution: FeCl₂•6H₂O, 3.78 g/L; Na₂MoO₄•H₂O, 0.7 g/L;ZnSO₄•7H₂O, 0.4 g/L; MnCl₂•4H₂O, 0.5 g/L; CuSO₄•5H₂O, 0.8 g/L;CoCl₂•6H₂O, 0.8 g/L; H₃BO₃, 0.2 g/L.

pH of each medium was maintained at about 7.0 using ammonium hydroxide.Temperature was controlled at about 32.0° C. and increased to about37.5° C. at induction. Feed medium and dextrose were sterilizedseparately and aseptically connected to a fermenter using siliconerubber tubing.

Supply of feed medium was initiated when the initial batch amount in theculture was depleted. A polarographic oxygen electrode monitoreddissolved oxygen tension in the fermenter. Carbon feed rate wascontrolled in a cascade mode to keep constant dissolved oxygen tensionabove about 25%. Aeration was maintained at about 10 slpm and thepressure at about 7.0 psig over pressure. Agitation speed of about 400rpm was used at the beginning and increased as required to maintainabout 25% dissolved oxygen tension. At an OD₅₅₀ of approximately 14units, 100 mL of 1.0 mM IPTG was injected into the culture, or thetemperature was increased to 37.5° C. to induce recombinant geneexpression.

None of the fermentation runs using a K-12 host background haddetectable 6-PGL adduct formation on recombinantly expressedpolypeptide, whereas runs using B strain in concert with T7 promoter allexhibited the presence of detectable 6-PGL adduct formation onrecombinantly expressed polypeptide. The three runs using B strain andthe pL promoter did not have detectable adduct formation. These datademonstrate that expressing a recombinant protein in a host strainhaving pgl activity, e.g. K-12, may prevent 6-PGL adduct formation inrecombinant proteins that are susceptible to this modification, seeExample 3. The presence of 6-PGL adduct formation in CXC chemokineexpressed in E. coli B and K-12 strains is presented for eachfermentation in Table 11.

TABLE 11 Presence of 6-PGL adduct on CXC chemokine expressed by T7 or pLpromoter in E. coli B and K-12 strains. Gluconolactone adduct 5 hr postinduction Strain genotype Promoter Run No. [% of product peak] B T7 121.7 2 5.8 3 4.2 Average 10.6 K-12 T7 1 0 2 0 Average 0 B pL 1 0 2 0 3 0Average 0 K12 pL 1 0 2 0 Average 0

Example 8

This example shows that expressing a heterologous polypeptide, humaninterleukin (human IL-18), in E. coli BL21 strain results in 6-PGLadduct formation, whereas expressing the same polypeptide in a K-12strain will prevent 6-phosphogluconoylation of internal lysines.

E. coli BL21 (DE3) and K-12 strains were constructed to express a humaninterleukin (IL-18). Cultures were grown in minimal medium as describedin Example. Monitoring for product modification with 6-PGL was conductedat various time points throughout the culture, using methods describedin Example.

Results are expressed as peak area ratios of unmodified product tomodified product. Higher numbers indicate a lesser proportion ofgluconoylated product. It is evident that the extent of gluconoylationin BL21 (DE3) strain is significantly greater than in K-12 strain.Phosphogluconoylation of a human interleukin (IL-18) expressed in B andK-12 strains of E. coli is summarized in Table 12.

TABLE 12 Phosphogluconoylation of a human interleukin expressed in B andK-12 strains of E. coli Area ratio of unmodified E. coli strain Timepost induction (h) product to modified product K-12 16 58.4 18 44.5 20N/d 22 29.3 24 30.0 BL21(DE3) 22 8.3 24 7.7 N/d = modified product notdetectable

Example 9

This example illustrates that overexpression of heterologous pgl in E.coli host cells leads to increased metabolic conversion of 6-PGL to itsproduct 6-phosphogluconate thereby reducing the pool of 6-PGL availablefor adduct formation. A P. aeruginosa pgl gene was cloned and engineeredinto a plasmid based inducible expression vector system that iscompatible with colE1 origin of replication based plasmids, such thattwo different plasmids may be maintained in the same host cell. Theinducible expression level of P. aeruginosa pgl can be regulated atwill, in a fashion independent of endogenous E. coli metabolic pathwaysand independent of the inducible expression of the recombinant proteinof interest. Accordingly, recombinant pgl expression can be regulatedsuch that the pool of 6-PGL was diminished below the level required foradduct formation, but not to the extent that host cell resources weresubstantially affected. Therefore, the level of the desired recombinantprotein can be separately regulated to give very high expression levels.

Cloning of P. aeruginosa pgl.

P. aeruginosa pgl was cloned as follows. P. aeruginosa was obtained fromthe American Type Culture Collection (American Type Culture Collection,12301 Parklawn Drive, Rockville Md. 20852-1776): Pseudomonas aeruginosa,strain ATCC 27853. 5′ and 3′ PCR amplification primers were designed andsynthesized to direct the PCR amplification of the entire pgl gene,including the region just upstream of the initiation codon ATG. Thesequence of the sense and anti-sense amplification primers are:

(SEQ ID NO: 4) 5′ GGCCTCGAGCTCGGTGGCCCTGGTGGCCC 3′ and (SEQ ID NO: 5) 5′GCCCTCGAGTCCGCCACTCAGGGGTACCAAT 3′respectively. Bolded sequences indicate XhoI sites introduced into thegene to enable subsequent cloning steps.

It was additionally found that the P. aeruginosa gene is very GC-richand will not PCR amplify under normal amplification conditions. It wasfound that PCR amplification requires the use of PCR amplificationconditions that are optimized for GC-rich templates, specificallyAdvantage-GC cDNA Polymerase Mix (cat. 8419-1; BD BiosciencesClontech:Palo Alto, Calif.; USA). The corresponding pgl DNA sequence(SEQ ID NO:7) encoding the polypeptide SEQ ID NO:8 was PCR amplifiedfrom whole P. aeruginosa cells after 25 cycles of 94° C. for 30 secondsfollowed by 68° C. for 1.5 minutes. The amplification was then completedby a single incubation at 68° C. for 3 minutes. The PCR product wasisolated by agarose gel electrophoresis and column chromatography over aQIAquick Gel Extraction Kit (cat. 28704, Qiagen: Valencia, Calif.; USA).The amplified, gel purified pgl DNA was eluted in water and immediatelycloned into the cloning vector pCR2.1 using a TOPO TA Cloning® kit (cat.K4550-40, Invitrogen: Carlsbad, Calif.; USA) and transformed into OneShots TOP10 Chemically Competent E. coli (cat. C4040-06, Invitrogen:Carlsbad, Calif.; USA). Resulting amp^(r) colonies were screened forinserts by EcoRI digestion and electrophoresis of the products. Sequenceverified inserts were released from the cloning vector by XhoI digestionand purified using the QIAquick Gel Extraction Kit. pECO-1 expressionvector DNA (BioCat 73552, GlaxoSmithKline: King of Prussia, Pa.; USA)was cut with SalI and treated with Calf Intestinal Alkaline Phosphatase(CIP) (cat M0290S, New England Biolabs:Beverly, Mass.; USA), and thenpurified using the QIAquick Gel Extraction Kit. The XhoI digestedinserts and the SalI treated pECO-1 were ligated using T4 DNA ligaseessentially as described by the manufacturer (cat. M0202S; New EnglandBiolabs: Beverly, Mass.; USA). Two microliters (2 μl) of the ligationreaction was transformed into One Shot® TOP10 chemically Competent E.coli cells as described by the manufacturer (cat. C4040-06; Invitrogen:Carlsbad, Calif.; USA). Colonies were screened for the presence pglinserts in the correct orientation by PCR. Clone pECO-Ipglcl2-13 wasselected for further study as set forth below.

The resulting construct is based on the pACYC184 plasmid backbone, andcomprises the following genetic functional elements: (1) p15A ori, whichallows plasmid episomal maintenance in the same host cell asheterologous plasmids with the colE1-type ori element; (2)chloramphenical^(r) selectable marker; (3) a tetracycline induciblepromoter upstream of a multiple cloning site; (4) the full length P.aeruginosa pgl gene.

The final construct, pECO-1 pglcl2-13, was transformed as describedabove into BL21 (DE3) cells expressing a human interleukin for furtherstudy. The DNA sequence of pECO-1pglcl2-13 sequence is presented as SEQID NO:9 and the restriction map of pECO-1 pglcl2-13 is presented in FIG.1.

Cultures were performed as described in Example 3. Analysis for 6-PGLadduct was performed as described in Example 4. Phosphogluconolactonaseactivity was measured as described in Example 6. Additional strainsexpressing the human interleukin (IL-18) that were examined in the studyinclude BL21 (DE3) without pECO-1 pglcl2-13 and K-12. The strains withthe pgl plasmid were examined with and without the addition ofanhydrotetracycline at 20 ng/mL. A summary of the presence of absence of6-PGL adduct formation in cells expressing pgl is summarized in Table 13as shown below.

TABLE 13 Impact of pgl expression on phosphogluconoylation of a humaninterleukin 6-PGL adduct Maximum pgl formation activity on humaninterleukin Strain IU/min/g DCW (IL-18) BL21 (DE3) 0.002 Present BL21(DE3) pECO-1pglcl2-13, 0.56 ± 0.13 Absent no tet BL21 (DE3)pECO-1pglcl2-13, 0.64 ± 0.11 Absent tet induced K-12 0.96 ± 0.14 AbsentDCW = dry cell weight

This example has demonstrated that co-expression of Pseudomonasphosphogluconolactonase with the human interleukin (IL-18) successfullyprevents formation of the 6-PGL adduct in the B strain of E. coli.

Example 10

Human interleukin-18 (IL-18) is an immunomodulatory cytokine that isbeing developed as an anti-cancer agent for the treatment of renal cellcarcinoma and malignant melanoma. Descriptions of human and murine IL-18are presented in the following U.S. patents: U.S. Pat. No. 6,582,689,U.S. Pat. No. 5,914,253, U.S. Pat. No. 5,879,942, U.S. Pat. No.5,912,324, U.S. Pat. No. 5,914,253, U.S. Pat. No. 6,060,283, U.S. Pat.No. 6,087,116, and U.S. Pat. No. 6,214,584, which are incorporatedherein by reference in their entirety. IL-18 is believed to stimulatethe immune system, promote Fas-induced tumor cell death and cellmediated immunity. The polypeptide sequence of IL-18 (SEQ ID NO:10) maybe produced in E. coli BL21(DE3). It may be expressed from thepolynucleotide sequence for pro-IL18 (SEQ ID NO:11), which is furtherprocessed to mature IL-18 by a processing enzyme, Caspase 5,co-expressed in the same cell.

6-Phosphogluconolactonase from Gram-negative bacterium, Pseudomonasaeruginosa, was cloned into a BL21 (DE3) strain. Production of IL-18 inthis strain produces IL-18 with no detectable adduct formation resultingin a direct increase in the yield of the desired product. Furthermore,the correction of the pgl deficiency has also resulted in >10% increasein specific productivity. Thus, the combined effect of product qualityand specific productivity improvements by the use of this modifiedproduction strain has translated into a >25% increase in titer yield atthe fermentation stage. It is anticipated that the elimination of adductformation will have a direct, positive impact on the purificationprocess and further increase the overall process yield.

Example 11

Gluconoylation of IL-18 was monitored by HPLC analyses on IL-18 producedin BL21 (DE3) cells with and without co-expression of pgl. Adductformation was detected as a shoulder peak eluting approximately twominutes after purified IL-18. When IL-18 was expressed in and purifiedfrom E. coli strain BL21DE3 (pgl minus), the main peak area (i.e., areaof the peak for IL-18 having no detectable gluconoyl adduct formation)for IL-18 was approximately 56%. While expression and purification ofIL-18 is BL21 (DE3) (pgl plus) cells produced a main peak area of about88% and showed no adduct formation based on the High Resolution HPLCassay.”

Example 12

Two vectors were created comprising polynucleotides that encodepro-IL-18 (SEQ ID NO: 11), Caspase 5 (SEQ ID NO:12). Both were pETvectors wherein polypeptide production was induced using a T7 promoter.One vector also comprised a polynucleotide encoding pgl from P.aeruginosa (SEQ ID NO:7) that was induced by the same promoter aspro-IL-18. The entire sequence of the pET vector comprising pro-IL-18and Caspase 5 is presented in SEQ ID NO:13. A restriction map of thisvector is presented in FIG. 2. The entire sequence of the pET vectorcomprising pro-IL-18, Caspase 5 and pgl is presented in SEQ ID NO:14. Arestriction map of this vector is presented in 3. When IL-18 wasexpressed in E. coli BL21 (DE3) cells using these vectors, the amount ofrecoverable IL-18 was greatly improved when the polypeptide wasco-expressed with pgl compared with the expression of IL-18 without pgl.The amount of recovered IL-18 obtained from each expression vector in E.coli BL21 (DE3) cells is presented in Table 14.

TABLE 14 IL-18 Maximum Titer Produced in “B” Strain E. coli with andwithout Co-expression of pgl Strain* pgl gene Maximum Titer (g/L) RBB057No 4.32 (+/−0.4) RBB059 Yes 6.78 (+/−0.1) *Both strains are E. coli Bstrain

All publications and references, including but not limited to patentsand patent applications, cited in this specification are hereinincorporated by reference in their entirety as if each individualpublication or reference were specifically and individually indicated tobe incorporated by reference herein as being fully set forth. Any patentapplication to which this application claims priority is alsoincorporated by reference herein in its entirety in the manner describedabove for publications and references.

1. A method for reducing gluconoylation of a heterologous polypeptideexpressed in E. coli, wherein said heterologous polypeptide is humanIL-18, said method comprising growing said E. coli in rich culturemedium, wherein the amount of gluconoylation on said human IL-18 isreduced as compared to E. coli grown in minimal medium.
 2. The method ofclaim 1, wherein the E. coli does not demonstrate significant6-phosphogluconolactonase activity when grown in minimal medium.
 3. Themethod of claim 1, wherein the E. coli is B strain.
 4. The method ofclaim 1, wherein the rich culture medium comprises a complex nitrogensource.
 5. The method of claim 4, wherein the rich culture medium iscapable of maintaining cell growth at ratio of 1:1 for concentration ofcomplex nitrogen source to cell density.
 6. The method of claim 4,wherein the complex nitrogen source comprises tryptone.
 7. The method ofclaim 4, wherein the complex nitrogen source comprises peptone.
 8. Themethod of claim 4, wherein the complex nitrogen source comprises yeastextract.
 9. The method of claim 1, wherein the culture medium isSuperbroth.
 10. The method of claim 9, wherein the Superbroth medium isdoubly concentrated.